1. Field of the Invention
The present invention relates to a method for producing heterodimer derivatives of protein phosphatase 2A (hereinafter abbreviated to as “PP2A”). More particularly, it relates to a method for production of a large quantity of heterodimer derivatives of PP2A consisting of the catalytic subunit and A subunit of PP2A, each carrying a tag, from which derivatives a highly pure PP2A can be prepared.
2. Description of the Related Art
PP2A is one of the most basic enzymes among hydrolases by which the phosphate groups attached to serine/threonine residues in a protein are hydrolyzed, and is formed as a complex of trimer in which a catalytic subunit termed PP2Ac is linked to an A subunit and a B subunit. Among these subunits, the catalytic subunit and the A subunit include two forms of α-isoform and β-isoform.
The PP2A which plays an important role in signal transduction in vivo is in big demand as a biochemical reagent and has come onto the market at a high price. In addition, in recent years, there is a need as a constitutive reagent for a convenient kit for measurement of toxic components, okadaic acids, accumulated in marine Bivalvia, or blue-green algal toxins (microcystins, nodularins), which are subjects to regulation as poisonous ingredients in lakes and marshes.
At present, as for PP2A used for the above-mentioned objects, no one but a heterodimer of the A subunit and the catalytic subunit has been known, which is referred to as PP2A and prepared from the human blood corpuscles.
The PP2A purified from the human blood corpuscles, however, is very expensive because of complicated purification process and difficulty of large-scale purification since it is separated from animal tissues. In addition, there was a problem in using the resulting PP2A as a biochemical reagent, since it is per se not likely to be sufficient in purity. Further, there was another problem that a large quantity of pure PP2A was unable to be produced at low cost or with easiness for the basic research, making it difficult to achieve research necessary for elucidation of a diversity of life process involving PP2A.
The present inventors previously studied a method for producing PP2A and found that in producing the intended catalytic subunit and A subunit of PP2A by a genetic engineering procedure using a baculovirus, attachment of a tag to the respective subunits allows easy separation from other proteins to obtain the subunits in a highly pure state. The inventors further found that production of the catalytic subunit and A subunit of PP2A carrying respectively different tags in the same cultured cells obtained a heterodimer of PP2A consisting of the catalytic subunit and A subunit respectively carrying different tags, and that use of these two tags possessed by these subunits allowed easy purification of the intended heterodimer only. A patent application was filed based on these findings.
When a baculoviral expression system was used, however, there was another problem that the rate of PP2A expressed as an insoluble protein was higher than that as a soluble protein, thereby decreasing the rate of PP2A to be purified, though the rate of expression was high.